The invention relates to a process for making fluorinated polymer adsorbent particles and to their use as a stationary phase for carrying out chromatographic separations.
Support materials for use in high productivity liquid chromatography must be mechanically strong in order to withstand operation at high rates of flow under high pressures. Moreover, they must be stable over the wide range of pH to which such materials are subjected during normal operation and regeneration. The stability of the polymeric particles in its environment allows it to withstand degradation and decomposition. Physical properties of particular importance to chromatographic media are (1) sphericity of the particles; (2) high surface area; (3) high pore volume and availability; (4) wide range of pore diameters; and (5) wide range of particle diameters.
The particles of the invention are an improvement over known particles in respect of many of the above properties. Furthermore, the fluorinated surface of certain of the particles of the invention present unusual and unexpected polarity that is beneficial in performing chromatographic separations such as that used for DNA.
The invention is therefore directed to the manufacture of improved fluorinated particles having adsorbent properties for superior performance as the stationary phase for use in chromatographic separations.
The invention provides a process for the preparation of porous spherical particles of fluorinated polymer adsorbent comprising the steps of:
(1) forming a water-insoluble solution of organic compounds comprising a monomer selected from C2-4 alkylene glycol esters of a C3-6 acrylic acid or divinyl benzene; a polyfluorinated vinyl monomer; a free radical initiator; and a water-insoluble, organic solvent-soluble porogenic material, the weight ratio of the comonomers to porogenic material being from 0.5:1 to 2:1;
(2) forming a dilute solution of a dispersing agent in water from which any oxygen has been purged with inert gas;
(3) with agitation and inert gas purging, rapidly dispersing the water-insoluble solution of organic compounds from step (1) into the dilute aqueous solution from step (2) and, as necessary, adjusting the temperature of the dispersion to 30-90xc2x0 C. to initiate copolymerization of the monomers, the level of mixing energy being sufficient to disperse the water-insoluble solution of organic compounds in the solution from step (2) in the form of liquid droplets having an average diameter of no more than 10-300 micrometers, at least 90% of the droplets being within 40% above or below the average mean particle diameter;
(4) continuing the agitation and oxygen purging of the dispersion from step (3) for a time sufficient to effect complete copolymerization of the monomers and particulation of the droplets in the form of finely divided polymer particles by precipitation of the copolymer therein;
(5) separating the finely divided copolymer particles from the polymerization reaction medium;
(6) extracting the porogenic material from the separated copolymer particles of step (5) by washing the particles with inert organic solvent, thereby forming pores within the copolymer; and
(7) drying the porous copolymer particles.
The invention further provides a process for the preparation of porous spherical particles of fluorinated polymer adsorbent comprising the steps:
(1) forming a water-insoluble solution of organic compounds comprising (a) a monomer selected from C2-4 alkylene glycol esters of a C3-6 acrylic acid and a divinyl benzene; (b) a polyfluorinated vinyl monomer; (c) a monomer selected from acrylic acid, methacrylic acid and esters thereof; (d) a free radical initiator; and (e) a water-insoluble, organic solvent-soluble porogenic material, the weight ratio of comonomers (a) plus (b) plus (c) to the porogenic material being from 0.5:1 to 2:1;
(2) forming a dilute solution of a dispersing agent in water from which any oxygen has been purged with inert gas;
(3) with agitation and inert gas purging rapidly dispersing the water-insoluble solution of organic compounds from step (1) into the dilute aqueous solution from step (2) and, as necessary, adjusting the temperature of the dispersion to 30-90xc2x0 C. to initiate copolymerization of the monomers, the level of mixing energy being sufficient to disperse the water-insoluble solution of organic compounds in the solution from step (2) in the form of liquid droplets having an average diameter of no more than 10-300 micrometers, at least 90% of the droplets being within 40% above or below the average mean particle diameter;
(4) continuing the agitation and oxygen purging of the dispersion from step (3) for a time sufficient to effect complete copolymerization of the monomers and particulation of the droplets in the form of finely divided polymer particles by precipitation of the copolymer therein;
(5) separating the finely divided copolymer particles from the polymerization reaction medium;
(6) extracting the porogenic material from the separated copolymer particles of step (5) by washing the particles with inert organic solvent, thereby forming pores within the copolymer; and
(7) drying the porous copolymer particles.
The invention further provides adsorbent particles made by the process described above.
The present invention further provides uses for the particles according to the invention as a stationary phase in chromatographic techniques. Certain particles of the invention are particularly suited to use where the sample to be chromatographed is a macromolecule containing nucleotides, nucleosides or polypeptides, such as DNA, RNA or endotoxins.
The invention relates to a method for making high quality adsorbent fluoropolymer particles by suspension polymerization with an aqueous solution containing a conventional dispersing agent. The basic components of the process are (1) the water-insoluble polymerization system, which is comprised mainly of a polyfluorinated monomer, two or more ethylenically unsaturated monomers and a free radical-initiating catalyst, and (2) the dispersion medium, which is a dilute aqueous solution containing a conventional dispersing agent. By water-insoluble solution, it is meant a solution sufficiently water-insoluble to permit suspension polymerization to occur. Preferred ethylenically unsaturated monomers are monomers having divinyl functionality. Non-fluorinated monomers having divinyl functionality are more preferred. Poly(vinyl alcohol) and poly(vinyl pyrrolidone) are preferred dispersing agents
A. Dispersing Agents
The polymerization of the polyfluorinated copolymer for use in the invention is conducted in the presence of a dilute aqueous solution containing a dispersing agent, for example poly(vinyl alcohol) or poly(vinyl pyrrolidone). The principal function of the dispersing agent is to adjust the interfacial surface tension between the finely dispersed water-insoluble polymerization components and the continuous aqueous medium phase. By regulating the concentration of dispersing agent dissolved in the aqueous medium, the droplet size of the dispersed polymerization system and thus the size of the resultant polymerized particles can be more finely controlled.
So long as the dispersing agent is essentially completely dissolved in the aqueous medium, a wide range of molecular weights of the dispersing agent may be used successfully in the practice of the invention. One preferred dispersing agent is PVA that is at least 80% hydrolyzed, and more preferably at least 86% hydrolyzed, with a molecular weight of at least about 1,000. The maximum usable molecular weight is a function of the ambient water solubility of the dispersing agent. For example, the molecular weight of the PVA used will ordinarily not exceed 150,000 and preferably is no higher than 100,000.
For the purposes of the invention, the concentration of PVA in the aqueous medium should be within the range of 1 to 50 mL PVA per liter of water. Below 1 mL/L the modifying effect of the PVA is insufficient and above about 50 mL/L no further advantage is discernible. It is, of course, desirable to use lesser amounts of PVA in order to avoid energy-wasting increases in viscosity of the aqueous medium.
B. Polymerization System
1. Polyfluorinated Monomer: As set out above, the fluorine-containing comonomer must contain a plurality of fluorine (F) substituents. It is preferred that the fluorinated comonomer contains at least three F substitutions. In addition to these restrictions on its degree of fluorination, it is essential that the fluorinated comonomer be essentially completely insoluble in water under the polymerization temperatures encountered and essentially completely soluble in the other components of the dispersed polymerization system.
Suitable polyfluorinated comonomers are those containing active vinyl sites such as acrylates, methacrylates, vinyl compounds, maleates and itaconates. Among the many compounds within those categories are pentafluorostyrene, bis-hexafluoroisopropyl itaconate, bis-hexafluoroisopropyl maleate, heptadecafluorodecyl acrylate, perfluorooctyl methacrylate, 2,2,3,3-tetrafluoropropyl methacrylate, mono-trifluoroethyl itaconate, 2,2,2-trifluoroethyl maleate, vinyl benzyl perfluoroctanoate and vinyl trifluoroacetate.
2. Vinyl Comonomers: It is preferred that the comonomer component of the polyfluorinated copolymer for use in the invention be a non-fluorinated C2-4 alkylene glycol ester of a C3-6 acrylic acid (the cross-linking comonomer). The cross-linking comonomer must have at least two vinyl groups. Suitable comonomers having this composition are ethyleneglycol dimethacrylate, 1,3-propyleneglycol dimethacrylate, 1,4-butanediol dimethacrylate, ethyleneglycol itaconate, ethyleneglycol diacrylate, and ethyleneglycol dimaleate. Divinyl benzene can also be used for this purpose.
A mixture of non-fluorinated comonomers can also be used, where one non-fluorinated comonomer has at least two vinyl groups, i.e. the cross-linking comonomer, and the third monomer, i.e. co-monomer (c), is acrylic acid, methacrylic acid, or an ester of acrylic or methacrylic acid. Typical esters are the methyl, ethyl, and hydroxyethyl esters of these acids, epoxide containing esters of these acids and amine esters of these acids. Thus, a forth co-monomers selected from co-monomers (c) may be used in the synthesis.
The presence of co-monomer (c) facilitates the attachment of ligands for use in chromatographic separations by obviating the use of PVA as a linker, as described in U.S. Pat. Nos. 5,773,587 and 6,046,246, between the perfluorinated particle and the ligand. The addition of monomer (c) has little effect on the properties of the improved particles of the invention, such as stability of the particle or pore size.
We have shown that between 1 and 30% of the cross-linker ethylene glycol dimethacrylate can be replaced with a third or forth monomer selected from co-monomers (c). These co-monomers can be chosen depending on the functionality desired. For example, functional esters of acrylic and methacrylic acid can be added such as those containing hydroxyl, epoxide, amine, quarternary ammonium, sulphonic acid etc. can be used.
3. Free Radical Initiator: An essential component of the polymerization is a source of free radicals. In particular, the system must contain one or more compounds that thermally decompose under the conditions of polymerization to form free radical species. A preferred free radial agent is a mixture of azo-bis-isobutyronitrile (AIBN) and benzoyl peroxide (BPO). From about 10 to about 50 mg/L are needed for this purpose. It is recognized that higher concentrations are operable functionally. However, it is preferred to use as small amounts as possible in order to lessen the amount of extraneous materials in the formed polymer particles.
C. Porogen
Suitable porogenic materials are those organic compounds which are (1) chemically inert with respect to the other components of the polymerization phase, (2) completely soluble in the polymerization system, (3) completely insoluble in the continuous aqueous phase and (4) readily extractable from the polymerized particles at relatively low temperatures with a low molecular weight organic solvent. Dibutyl phthalate, which is easily removed by washing the polymer particles with dichloromethane, is a preferred porogen for use in the invention. Other suitable porogens include toluene, isopropyl benzene, 2-methyl-4-pentanone, 2-methyl-4-pentanol and chlorobenzene.
D. Polymerization Procedure
The polymerization should be conducted in the essentially complete absence of air or any other source of oxygen contamination, which might lead to adverse reactions with any of the components of the polymerization system, especially the monomers, crosslinking agent and free radical initiator. It has been found that the most practical way of removing and preventing the introduction of oxygen into the polymerization system is continuously to purge the polymerization reaction system before, during, and after completion of the polymerization process with an inert gas. Any of the inert gases are, of course, suitable for this purpose. However, argon and nitrogen are the least expensive and will be preferred in most instances. Because the polymerization is conducted under very high energy mixing conditions, the method of introducing the purging gas is not particularly critical, so long as it is adequate in volume.
The dispersing agent functions principally for more precise control of interfacial tension between the dispersed monomer droplets and the aqueous continuous medium. The droplet size is controlled more dominantly by the amount of mixing energy used to disperse the polymerization system. Thus, only comparatively low concentrations of PVA as dispersing agent are required in the aqueous medium, e.g., on the order of 1-100 g/L. A PVA concentration within the range of 0.5-40 g/L is preferred. Though higher concentrations can be used, they do not improve functionality. Because of the necessity of forming very small droplets during the polymerization, it is, of course, desirable to avoid higher PVA concentrations which would render the aqueous medium more viscous.
The amount of energy input into the polymerization is primarily a function of the polymer particle size that is desired. Thus, if larger particles are sought, the degree of mixing (energy input) is lowered. If smaller particles are sought, the degree of mixing is raised. It is preferred that droplet size during polymerization be controlled to obtain polymer particles within the range of 5-300 micrometers, 20-100 micrometers being especially preferred.
F. Particle Properties
Ideal chromatography media need to have the following properties: (1) spherical shape; (2) high surface area; availability of a wide range of (3) pore diameters and (4) particle diameters; (5) high pore volume; (6) high mechanical strength; and (7) both chemical and mechanical stability throughout the pH range to which the media are exposed in use.
Sphericity of the particles, rather than irregular, granular shapes, is advantageous for providing minimum resistance to flow through a packed bed of the particles and minimum backpressure. Such regularly shaped particles are less likely to undergo densification during use.
Particle size and size distribution are also important properties of the particles of the invention. In general, particles larger than about 20 micrometers facilitate lower backpressure in packed columns. Moreover, the chromatographic peak width and peak shape obtained with larger particles are usually wider than the peak width and shape obtained with particles in the range of 3-15 micrometers. Narrow peak shapes are frequently desired for many types of separations.
The available surface area of polyfluorinated particles produced by the method of the invention is ordinarily preferred to be at least about 200 m2/g in order to obtain higher loading of antigens on the particulate media. Nevertheless, media having much lower surface areas can readily be made according to the invention by changing the amount of porogen used in the polymerization system and decreasing the size of the particles. Concomitantly, a large pore volume of at least 0.5 mL/g is needed in order to obtain a high surface area.
A wide range of pore sizes must be available for different chromatographic procedures. Large pores are needed for the efficient capture of larger molecules, such as proteins, while small pores are needed for the efficient capture of small molecules. In general, the range of pore sizes may extend from below 60 xc3x85 to as high as 1,000 xc3x85, 300-800 xc3x85 being preferred. This range of sizes is quite readily available using the invention method of adjusting the relative amount and type of porogen within the formed polymer particles.
Because of the wide range of pH values at which chromatography media are used and because of the very high pH ranges that are encountered frequently to clean and regenerate them, it is necessary that they be chemically inert throughout the entire range of such pH exposures. In particular, chromatographic media must be able to withstand the high pH (12 or higher) encountered by the use of NaOH for cleaning the media particles, typically 0.1-1 normal.
G. Uses of the Particles
The adsorbent particles of the invention are quite versatile and may be used as the stationary phase for carrying out a wide variety of chromatographic separations. Examples of the chromatographic separations contemplated include reverse phase separations, affinity separations, expanded bed separations, ion-exchange chromatography, gel filtration, chromatographic component separation, solid phase extraction, filtration and other recognised technical methods of distinguishing, measuring or collecting components of a chemical, biological or physical mixture. The particles may be used as support for grafting different types of ligands. Certain of the particles are particularly suited to use where the sample to be chromatographed is DNA, RNA or polypeptides.
The polyfluorinated particles of the invention can be used for chromatographic separations either with or without a coating of a hydrophilic polymer, such as poly(vinyl alcohol).
The surface of the uncoated particles of Examples 3 and 4 is hydrophobic, but with a slight polarity, which combination of properties is ideal for reverse phase chromatographic separations. Reverse phase chromatography involves the use of a relatively non-polar stationary phase in conjunction with a very polar mobile phase that is usually water. This technique is used to separate solutes of lower polarity. Reverse phase chromatography is usually performed using silica that is coated with an organic silane to provide hydrophobicity. However, the hydrophobized silica has a severe limitation in that it cannot be used at pH greater than 11 and cannot be cleaned with concentrated caustic soda solutions without dissolving the particles. A substantial advantage of the polyfluorinated particles of the invention is that they do not have this limitation.
The use of the uncoated invention particles for reverse phase chromatography is illustrated by Example 28 and the stability of the particles of the invention toward basic solutions is shown by the data obtained in Example 29 below.
Suitable hydrophilic polymers for use in coating the polyfluorinated particles of the invention are those which are uncharged, water-soluble, non-cyclic and have a multiplicity of hydroxyl groups. Though many several such hydrophilic polymers are useful for this particular function, (polyvinyl alcohol) is preferred.
Advantageously, the polyfluorinated compounds of the invention may be used in medical devices with or without ligands on their surfaces to do separations that are not classified as chromatographic. For example, components of blood can be separated using a medical device in which the blood is pumped through a cartridge extra-corporeally and returned to the body. A component such as a toxin would be removed and not returned to the body.
Due to the stability of the polyfluorinated particles of the invention, sterilization can be done by gamma irradiation without destroying the particle. This property makes the particles particularly well suited for uses in medical devices that must be sanitized.
E. Derivatization of Particles
If desired, the PVA-coated polyfluorinated particles of Examples 5 and 6 can be functionalized by reacting suitable molecules with the hydroxyl groups of the PVA. Thus, strong cationic ion exchange functionality can be provided to the particle surfaces by placing sulfonic acid groups on the surface. Likewise, strong anionic ion exchange functionality can be provided by applying quaternary amines. Weak cation functionality can be produced by the use of carboxylic groups and weak anion functionality can be obtained by the use of primary amines.